Modified flagellin improved toll-like receptor 5 stimulating activity

ABSTRACT

Disclosed herein are flagellin mutants having an enhanced activity of stimulating the toll-like receptor-5 (hereinafter referred to as “TLR5”). More specifically, disclosed are flagellin mutants, prepared by point-mutating some of the amino acids of a TRL5 agonist flagellin so as to enhance the TRL-stimulating activity of the flagellin.

TECHNICAL FIELD

The present invention relates to flagellin mutants having an enhancedactivity of stimulating the toll-like receptor-5 (hereinafter referredto as “TLR5”), and more particularly to flagellin mutants, prepared bypoint-mutating some of the amino acids of a TRL5 agonist flagellin so asto enhance the TRL5-stimulating activity of the flagellin.

BACKGROUND ART

Flagella are important structural elements determining the motility ofbacteria and are generally composed of a hook, a basal body and afilament. It is known that flagella contribute to the swimming orswarming motility, the taxis of bacteria, the adhesion of pathogenicmicroorganisms to host cells, and formation of biofilms. A subunitprotein forming the flagellar filament is referred as flagellin, andflagellins are regularly assembled to form the filament. Hayashi et al.reported that TLR5 expressed on mammalian cells recognize the flagellinof gram-negative and gram-positive bacteria to activate NF-KB (HayashiF, Smith K D, Ozinsky A, Hawn T R, Yi E C, Goodlett D R, Eng J K, AkiraS, Underhill D M, Aderem A: Nature 410:1099-1103, 2001).

Toll-like receptors (TLRs) are typical “Pattern Recognition Receptors(PRRs)”, which recognize “Pathogen Associated Molecular Patterns(PAMPs)” present in pathogens and are found not only in mammals, butalso on the surfaces of insects and plant cells. Thirteen kinds of TLRshave been found to date, and studies on agonists of TLRs have beenactively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124(4):783-801, 2006).

PRRs such as TLRs are distributed on the surface or in the cytoplasm ofhost cells, induce innate immune responses after being stimulate withvarious PAMPs, and furthermore, regulate adaptive immune responses.Thus, TLR agonists can serve targets suitable for the development ofvarious immune regulators, particularly vaccine adjuvants.

As used herein, the term “vaccine adjuvants” refers to substances whichcan enhance, prolong or accelerate Ag-specific immune responses inducedby vaccine antigens, when they are co-administered with vaccines.Vaccine adjuvants approved for use in the human body include aluminumphosphate, aluminum hydroxide, and squalene emulsion. Vaccine adjuvantsmust satisfy at least one of the following five requirements: 1)regulation of expression of co-stimulatory molecules on the surface ofantigen-presenting cells, induction of antigen-specific T-lymphocyteresponses, or immunomodulation such as the modulation of cytokinesecretion; 2) antigen presentation; 3) induction of CD8+ cytotoxic Tlymphocyte responses; 4) targeting; and 5) depot generation.

Ideal vaccine adjuvants: 1) must be safe; 2) must be biodegraded invivo; 3) must show potent protective or therapeutic immune responsescompared to when antigens are administered alone; 4) must be chemicallyor biologically verified substances; 5) preferably act at concentrationslower than antigens; and 6) must have a long half life, such that theycan be readily applied commercially or clinically.

Substances, which are currently used as vaccine adjuvants or consideredfor use as vaccine adjuvants, include: 1) mineral salts such as aluminumhydroxide gel; 2) surfactant substances; 3) bacteria-derived substances;4) cytokines or hormones; 5) polyanions; 6) polyacryl; 7) carriers; 8)living vectors comprising virus; and 9) vehicles, such as mineral oilliposomes. Among them, protein-derived vaccine adjuvants, which arecurrently actively studied and receive great attention, include Vibriocholerae-derived cholera toxin (CT) and Escherichia coli-derivedheat-labile toxin (LT). It was reported that these vaccine adjuvantsinduce the production of antigen-specific antibodies in mucosal areasand sera and induce the expression of B7-2 on the surface ofantigen-presenting cells (APCs) to stimulate the co-stimulatorysignaling of CD4+ helper T cells. However, these adjuvants are exotoxinshaving high enterotoxicity, and thus studies focused on reducing thetoxicity thereof and increasing the adjuvanticity thereof, are inprogress.

As disclosed in PCT International Patent Publication No. WO 2005/070455,the present inventors constructed a transposon mutant library in orderto screen adhesion and invasion factors of Vibrio vulnificus, andanalyzed the Tn-flanking regions in three clones, which lost theiradhesion to host cells and their motility, and, as a result, the presentinventors identified two flagella operons consisting of 56 genes. Inthis analysis process, it was found that V. vulnificus has a total ofsix flagellin genes (flaA, flaB, flaF, flaC, flaD and flaE), and amongthem, the flaB gene is the major component of the flagellins. Thepresent inventors studied the possibility that the flagellin recombinantprotein (FlaB), the component of the polar flagellin of V. vulnificus,can be applied as a component vaccine against V. vulnificus, and, as aresult, the present inventors found that the flagellin recombinantprotein (FlaB), besides high antigenicity, also has a strong vaccineadjuvant effect. To elucidate this finding, the present inventorsconducted further studies. As a result, it was demonstrated that, when avaccine antigen tetanus toxoid was administered to the nasal cavity oftest animals together with flagellin, the flagellin amplified the effectof the vaccine by transmitting a signal to the TLR5 of host cells toactivate the immune system, and when a lethal dose of tetanus toxin waschallenged to mice immunized by administering tetanus toxoid andflagellin, the flagellin induced complete defense immunity to the toxin,suggesting that the flagellin had an excellent mucosal vaccine adjuvanteffect (Lee S E, Kim S Y, Jeong B C, Kim Y R, Bae S J, Ahn O S, Lee J J,Song H C, Kim J M, Choy H E, Chung S S, Kweon M N, Rhee J H.: Infect.Immun. 74: 694-702, 2006).

Among various TLR agonists, flagellin stimulating TLR5 is a proteincomponent, unlike other TLR agonists [CpG-DNA, MLP (mycoplasmallipopeptide)]. Thus, it is possible to synthesize recombinant flagellinproteins, the quality of which can be continuously controlled, and inaddition, it is possible to construct various recombinant fusionproteins having an enhanced activity of stimulating TLR5.

According to the results of studies on the three-dimensional structureof flagellin, polar amino acid residues and charged amino acid residuesin a flagellin monomer react with those in another monomer, so that theaxial interaction between the monomers occurs to form a polymer, thusforming the characteristic filament structure of flagellin (Yonekura K,Maki-Yonekura S, Namba K: Complete atomic model of the bacterialflagellar filament by electron cryomicroscopy. Nature. 2003424(6949):643-50; Samatey F A, Imada K, Nagashima S, Vonderviszt F,Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellarprotofilament and implications for a switch for supercoiling. Nature.2001 410(6826):331-7). According to the study of Smith et al., it wasreported that TLR5 does not recognize a filament-type flagellin polymer,but recognizes flagellin monomers (Smith K D, Andersen-Nissen E, HayashiF, Strobe K, Bergman M A, Barrett S L, Cookson B T, Aderem A. Toll-likereceptor 5 recognizes a conserved site on flagellin required forprotofilament formation and bacterial motility. Nat. Immunol. 20034(12):1247-53).

A common problem, observed in preventive and therapeutic vaccines(vaccines for infectious diseases, autoimmune diseases, allergicdiseases and cancers), which are currently used in clinicalapplications, is that these vaccines lack effective vaccine adjuvants,which can specifically amplify relevant responses. Thus, there is astrong need to develop safer and more effective vaccine adjuvants.

DISCLOSURE Technical Problem

Accordingly, the present inventor have prepared recombinant flagellinmutants, which can suppress polar-charge reactions that are involved inthe axial interaction between flagellin monomers, of the flagellin geneflaB of V. vulnificus, by changing amino acid residues anticipated to beinvolved in the axial interaction, and have found that the preparedflagellin mutants have significantly enhanced TLR5-stimulating activitycompared to that of prior (? wild type?) flagellin proteins, therebycompleting the present invention.

Therefore, it is an object of the present invention to provide flagellinmutants having an enhanced activity of stimulating TLR5.

It is another object of the present invention to provide a vaccineadjuvant containing at least one of said flagellin mutants as an activeingredient.

Technical Solution

To achieve the above objects, the present invention provides flagellinmutants for suppressing the interaction between flagellin monomers inthe TLR5 agonist flagellin.

Also, the present invention provides a vaccine adjuvant containing atleast one of said flagellin mutants as an active ingredient.

ADVANTAGEOUS EFFECTS

In the present invention, more improved flagellin vaccine adjuvants weredeveloped by providing flagellin mutants having enhanced TLR-stimulatingactivity compared to that of a prior flagellin, which was found to showa potent mucosal vaccine adjuvant effect by stimulating TLR5 asdisclosed in PCT International Patent Publication No. WO 2005/070455.The flagellin mutants according to the present invention will be appliedfor the treatment of infectious diseases, autoimmune diseases, allergicdiseases and the like, and furthermore, in anticancer therapy, and willprovide an important ring connecting basic research with bedsideclinical studies. Thus, the application of the inventive flagellinmutants to a variety of vaccine formulations can create very highadded-value.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the comparison of amino acid homology between theSalmonella flagellin St-FliC, the E. coli flagellin Ec-FliC and the V.vulnificus flagellin Vv-FlaB, in which the domain of the flagellinprotein of each bacterial strain is indicated as a box (Samatey F A, etal., Structure of the bacterial flagellar protofilament and implicationsfor a switch for supercoiling. Nature. 2001 410(6826):331-7).

FIG. 2 is a schematic diagram showing a strategy inducing site-directedmutations in of the V. vulnificus flagellin gene flaB in theconstruction of the flagellin mutant according to the present invention(Samatey F A, et al., Structure of the bacterial flagellar protofilamentand implications for a switch for supercoiling. Nature. 2001410(6826):331-7).

FIGS. 3 and 4 show the effect of the inventive recombinant flagellinmutant on flagellin polymerization in an aqueous solution.

FIGS. 5 and 6 are graphic diagrams showing the measurement results ofTLR-stimulating activity of the flagellin mutant according to thepresent invention.

BEST MODE

Hereinafter, the present invention will be described in further detail.

The present invention relates to flagellin mutants, prepared bypoint-mutating some of the amino acids of a TLR5 agonist flagellin, suchthat flagellin mutation suppress the multimerization of flagellinmonomers, thus showing an enhanced activity of stimulating TLR5.

The present invention relates to: a flagellin mutant (D70A) of SEQ IDNO: 2, prepared by site-directed mutagenesis of aspartic acid (D70) toalanine (A) at position 70 of the wild-type V. vulnificus flagellin FlaBanalyzed to be involved in the axial interaction between flagellinmonomers in the TLR5 agonist flagellin; a flagellin mutant (R93A) of SEQID NO: 4, prepared by site-directed mutagenesis of arginine (R93) toalanine (A) at position 93; a flagellin mutant (L97W) of SEQ ID NO: 6,prepared by site-directed mutagenesis of leucine (L97) to tryptophan (W)at position 97; a flagellin mutant (N101A) of SEQ ID NO: 8, prepared bysite-directed mutagenesis of asparagine (N101) to alanine (A) atposition 101; a flagellin mutant (S103W) of SEQ ID NO: 10, prepared bysite-directed mutagenesis of serine (S103) to tryptophan (W) at position103; a flagellin mutant (E108A) of SEQ ID NO: 12, prepared bysite-directed mutagenesis of glutamic acid (E108) to alanine (A) atposition 108; a flagellin mutant (N135D) of SEQ ID NO: 14, prepared bysite-directed mutagenesis of asparagine (N135) to aspartic acid (D) atposition 135; a flagellin mutant (A151W) of SEQ ID NO: 16, prepared bysite-directed mutagenesis of alanine (A151) to tryptophan (W) atposition 151; a flagellin mutant (N153W) of SEQ ID NO: 18, prepared bysite-directed mutagenesis of aspargine (N153) to tryptophan (W) atposition 153; and a flagellin mutant (V291W) of SEQ ID NO: 20, preparedby site-directed mutagenesis of valine (V291) to tryptophan (W) atposition 291. Also, among the flagellin mutants, flagellin mutantshaving two or more amino acid substitutions fall within the scope of thepresent invention, and examples thereof may include a flagellin mutant(D70A/N135D) of SEQ ID NO: 44, prepared by site-directed mutagenesis ofaspartic acid (D70) to alanine (A) at position 70 of the wild-type V.vulnificus flagellin FlaB, and asparagine (N135) to aspartic acid (D) atposition 135, but the scope of the present invention is not limitedthereto.

As used herein, the term “flagellins” refers to single moleculesconstituting flagella that determine the mobility of bacteria.

The flagellin mutants, provided according to the present invention, havea significantly enhanced activity of stimulating TLR5, and thisphenomenon is because the flagellin mutants significantly reduceflagellin multimerization compared to prior flagellin mutants, thussuppressing flagellin polymerization.

Accordingly, the flagellin mutants having significantly enhancedTLR5-stimulating activity, prepared according to the present invention,can provide more potent TLR agonists as substitutes for the priorflagellins, thus providing vaccine adjuvants having increased efficiencycompared to that of the prior flagellins.

A process of demonstrating the enhanced TLR5-stimulating activity of theflagellin mutants according to the present invention comprises the stepsof:

1) preparing flagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20 and 44;

2) confirming that the flagellin mutants suppress flagellinpolymerization in an aqueous solution; and

3) confirming the TLR5 activation of each of recombinant flagellinmutants induced in E. coli.

[Mode for Invention]

Hereinafter, the present invention will be described in further detailwith reference to examples and test examples. It is to be understood,however, that these examples and test examples are illustrative only,and the scope of the present invention is not limited thereto.

The characteristics of strains and plasmids, used in the presentinvention, are summarized in Table 1 below. A method of preparing eachof flagellin mutants, and the detailed characteristics of each flagellinmutant, are shown in examples and test examples.

TABLE 1 Strains or plasmids Characteristics Sources Escherichia coliDH5α F recA1; restriction negative Our laboratory ER2566 F-λ-fhuA2[lon]ompT lacZ::T7 gene1 New England gal sulA11 (mcrC-mrr)114::IS10R Biolab(mcr-73::miniTn10-TetS)2 R(zgb-210::Tn10)(TetS) endA1 [dcm] BL21(DE3)F-ompT hsdS_(B()r_(B) − m_(B)−) Novagen gal dcm (DE3) Plasmids pCR2.1TOPO PCR TA cloning vector, Amp^(R), Km^(R) Invitrogen pTYB12 IMPACT(Intein Mediated Purification New England with an AffinityChitin-binding Tag) Biolab expression vector, Amp^(R)

<Culture and Storage of Each Strain>

E. coli strains, used in the following examples and test examples, werecultured in Luria Bertani media (Difco Co.). The cultured strains werestored in 30% glycerol in a freezer at −80° C.

Example 1 Preparation of Recombinant Flagellin Mutants by Site-DirectedMutagenesis

According to the previously reported data of analysis for thethree-dimensional structure of Salmonella flagellin (Yonekura K,Maki-Yonekura S, Namba K: Complete atomic model of the bacterialflagellar filament by electron cryomicroscopy. Nature. 2003424(6949):643-50; Samatey F A, Imada K, Nagashima S, Vonderviszt F,Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellarprotofilament and implications for a switch for supercoiling. Nature.2001 410(6826):331-7), and the bioinformatics analysis conducted by thepresent inventors, amino acid residues required for the axialinteraction between V. vulnificus flagellin FlaB monomers are asfollows. That is, it was analyzed that aspartic acid (D70) at position70 of the upper subunit of each monomer, which is involved in the axialinteraction, asparagine (D135) at position 135 of the upper subunit, andalanine (A151) at position 153 of the upper subunit, form multimersthrough polar-charge reactions with asparagines (N100) at position 100of the lower subunit of each monomer, glutamic acid (E108) at position108 of the lower subunit, arginine (R93) at position 93 of the lowersubunit and alanine (A292) at position of the lower subunit,respectively. That is, the polar-charge reactions occur between thefollowing amino acid residues: upper flagellin D70 and lower flagellinN100; upper flagellin N135 and lower flagellin E107; upper flagellinA151 and lower flagellin R93; and upper flagellin N153 and lowerflagellin A292.

In the present invention, the following flagellin mutants were preparedby site-directed mutagenesis of the following amino acid residuesanalyzed to be involved in the axial interaction between flagellinsubunits: a flagellin mutant (D70A) of SEQ ID NO: 2, prepared bysite-directed mutagenesis of aspartic acid (D70) to alanine (A) atposition 70; a flagellin mutant (R93A) of SEQ ID NO: 4, prepared bysite-directed mutagenesis of arginine (R93) to alanine (A) at position93; a flagellin mutant (N101A) of SEQ ID NO: 8, prepared bysite-directed mutagenesis of asparagine (N101) to alanine (A) atposition 101; a flagellin mutant (E108A) of SEQ ID NO: 12, prepared bysite-directed mutagenesis of glutamic acid (E108) to alanine (A) atposition 108; a flagellin mutant (N135D) of SEQ ID NO: 14, prepared bysite-directed mutagenesis of asparagine (N135) to aspartic acid (D) atposition 135; a flagellin mutant (A151W) of SEQ ID NO: 16, prepared bysite-directed mutagenesis of alanine (A151) to tryptophan (W) atposition 151; and a flagellin mutant (N153W) of SEQ ID NO: 18, preparedby site-directed mutagenesis of aspargine (N153) to tryptophan (W) atposition 153. However, alanine (A292) at position 292, analyzed to beinvolved in the axial interaction between flagellin monomers, wasexcluded from the construction of mutants, because it was thought to notbe changed even after it would be mutated.

In addition, the following mutants were prepared by site-directedmutagenesis of the following amino acid residues inferred to be involvedin the axial interaction between flagellin monomers: a flagellin mutant(L97W) of SEQ ID NO: 6, prepared by site-directed mutagenesis of leucine(L97) to tryptophan (W) at position 97; a flagellin mutant (S103W) ofSEQ ID NO: 10, prepared by site-directed mutagenesis of serine (S103) totryptophan (W) at position 103; and a flagellin mutant (V291W) of SEQ IDNO: 20, prepared by site-directed mutagenesis of valine (V291) totryptophan (W) at position 291.

First, in order to obtain template DNA for site-directed mutagenesis, a1,142-bp DNA fragment containing the flaB gene ORF was amplified by PCRusing a PCR primer FlaB-V5 of SEQ ID NO: 21(5′-gcggccgcatggcagtgaatgtaaatgtaaatacaaac-3′; underlined portion: NotIrestriction enzyme recognition site for cloning) and a PCR primerFlaB-V4 of SEQ ID NO: 22 (5′-cccggggcctagtagacttagcgctga-3′; underlinedportion: SmaI restriction enzyme recognition site for cloning. The PCRamplification was performed using the primers FlaB-V5 and FlaB-V4 in thefollowing conditions: initial denaturation at 95° C. for 1 min, and then30 cycles of denaturation at 95° C. for 30 sec, annealing at 70° C. for30 sec and extension at 72° C. for 1 min, followed by final extension at72° C. for 10 min. The amplified flaB DNA fragment was cloned into a PCR2.1-TOPO cloning vector (Invitrogen Co.), and the resulting vector wasnamed “pCMM270”. The pCMM270 plasmid was used as template DNA forsubsequent site-directed mutagenesis.

PCR conditions and cloned plasmids for the construction of site-directedmutants of the V. vulnificus flagellin gene flaB are summarized in Table2 below.

TABLE 2 Name of Name of Mutation PCR annealing pCR2.1-TOPO pTYB12position Primer (SEQ ID NO) temperature clone clone D70AFlaB-SD1 (SEQ ID NO: 23) 63° C. pCMM272 pCMM2765′-gtacgtaacgccaacgcaggtatctcaatc-3′ FlaB-SD2 (SEQ ID NO: 24)5′-gattgagatacctgcgttggcgttacgtac-3′ R93A FlaB-SD13-2 (SEQ ID NO: 25)63° C. pCMM281 pCM291 5′-catcctacaacgtatggctgacctatctctacaatc-3′FlaB-SD14-2 (SEQ ID NO: 26) 5′-gattgtagagataggtcagccatacgttgtaggatg-3′L97W FlaB-SD17 (SEQ ID NO: 27) 66° C. pCMM282 pCMM2925′-gcgtgacctatcttggcaatccgcgaacgg-3′ FlaB-SD18 (SEQ ID NO: 28)5′-ccgttcgcggattgccaagataggtcacgc-3′ N101A FlaB-SD9-3 (SEQ ID NO: 29)64° C. pCMM283 pCMM293 5′-ctacaatccgcggccggctcaaactcaaaatc-3′FlaB-SD10-3 (SEQ ID NO: 30) 5′-gattttgagtttgagccggccgcggattgtag-3′ S103WFlaB-SD15 (SEQ ID NO: 31) 63° C. pCMM284 pCMM2945′-caatccgcgaacggctggaactcaaaatcag-3′ FlaB-SD16 (SEQ ID NO: 32)5′-ctgattttgagttccagccgttcgcggattg-3′ E108A FlaB-SD11 (SEQ ID NO: 33)63° C. pCMM285 pCMM295 5′-caaaactcaaaatcagcgcgcgtggcgattc-3′FlaB-SD12 (SEQ ID NO: 34) 5′-gaatcgccacgcgcgctgattttgagtttg-3′ N135DFlaB-SD3 (SEQ ID NO: 35) 63° C. pCMM273 pCMM2775′-cgtcttttggtggtgacaagctgctaaacg-3′ FlaB-SD4 (SEQ ID NO: 36)5′-cgtttagcagcttgtcaccaccaaaagacg-3′ A151W FlaB-SD5 (SEQ ID NO: 37) 63°C. pCMM286 pCMM296 5′-gcaatgcaaattggttgggataacggtgaagcg-3′FlaB-SD6 (SEQ ID NO: 38) 5′-cgcttcaccgttatcccaaccaatttgcattgc-3′ N153WFlaB-SD7 (SEQ ID NO: 39) 67° C. pCMM287 pCMM2975′-caaattggtgcggattggggtgaagcggtcatg-3′ FlaB-SD8 (

 40) 5′-catgaccgcttcaccccaatccgcaccaatttg-3′ V291WFlaB-SD19 (SEQ ID NO: 41) 67° C. pCMM288 pCMM2985′-gcgcaagagtcgtgggcgattgtggatgcg-3′ FlaB-SD20 (SEQ ID NO: 42)5′-cgcatccacaatcgcccacgactcttgcgc-3′ D70A/N135D FlaB-SD1 (SEQ ID NO: 23)63° C. pCMM274 pCMM278 5′-gtacgtaacg ccaacgcagg tatctcaatc-3′FlaB-SD2 (SEQ ID NO: 23) 5′-gattgagata cctgcgttgg cgttacgtac-3′

In order to construct each of the site-directed mutant flaB DNAs (D70A,R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W andD70A/N135D), a mutant strand was synthesized by PCR reaction using eachof the primer pairs shown in Table 2 above, pCMM270 as a PCR template,and a proofreading Pfu polymerase in accordance with the manufacturer'sinstruction (Stratagene Co.). Each of the PCR reactions was performed inthe following conditions: initial denaturation at 95° C. for 45 sec, andthen 16 cycles of denaturation at 95° C. for 45 sec, annealing for 1 minat the temperature shown in Table 2, and extension at 68° C. for 10 min,followed by final extension at 68° C. for 10 min. Each of the amplifiedsite-directed mutant flaB DNAs of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17 and 19 was cloned into a PCR 2.1-TOPO cloning vector, and theresulting vectors were named “pCMM272”, “pCMM281”, “pCMM282”, “pCMM283”,“pCMM284”, “pCMM285”, “pCMM273”, “pCMM286”, “pCMM287” and “pCMM288”(Table 2).

To prepare a D70A/N135D dual site-directed mutant flagellin D70A/N135Dof SEQ ID NO: 44, pCMM273 with flaB DNA was used as a template for PCRreaction. The mutant strand (D70A) was synthesized by PCR using a PCRprimer FlaB-SD1 of SEQ ID NO: 23, a PCR primer FlaB-SD2 of SEQ ID NO: 24and a proofreading Pfu polymerase in accordance with the manufacturer'sinstruction (Stratagene Co.). The PCR reaction was performed in thefollowing condition: initial denaturation at 95° C. for 45 sec, and then16 cycles of denaturation at 95° C. for 45 sec, annealing at 63° C. for1 min and extension at 68° C. for 10 min, followed by final extension at68° C. for 10 min. The amplified D70A/N135D dual site-directed mutantflaB DNA of SEQ ID NO: 43 was named “pCMM274”.

Each of the DNA fragments of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17and 19, containing the site-directed mutant flaB genes (D70A, R93A,L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D),were digested with restriction enzymes NotI and SmaI, and then clonedinto a pTYB12 plasmid (New England Biolabs) digested with the samerestriction enzymes. The resulting plasmids were named “pCMM276”,“pCMM291”, “pCMM292”, “pCMM293”, “pCMM293”, “pCMM294”, “pCMM295”,“pCMM277”, “pCMM296”, “pCMM297”, “pCMM298” and “pCMM278”, respectively.These plasmids were electroporated into E. coli ER2566, and 0.5 mM of5-bromo-indole-3-chloro-isopropyl-β-D-glactopyranoside (IPTG) was addedthereto to induce the expression of the mutant flagellin genes. Pointmutant flagellin (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W,N153W, V291W and D70A/N135D) proteins were obtained from intein fusionproteins using 1,4-dithiothreitol (1,4-DTT) and a chitin bead columnaccording to the manufacturer's instruction. Before use, endotoxincontained in the isolated point mutant proteins was removed usingAffinityPak™ Detoxi Gel™ Endotoxin Removing gel (Pirece). The isolatedrecombinant proteins were subjected to gel filtration using a Superdex120 column (AKTA-Prime, Amersham), thus purifying the recombinantflagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and44 with high purity.

Test Example 1 Production of Recombinant Flagellin Mutants andBiochemical Properties Thereof in Aqueous Solution

In order to prepare recombinant flagellin mutants, each of the genes ofSEQ ID NOS: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 43,encoding the flagellin mutants, was cloned into a pTYB12 plasmid, andthen transfected into E. coli ER2566. 0.3 mM IPTG(isopropyl-β-D-1-thiogalactoside) was added to the media to induce theproduction of recombinant flagellin mutants, and then, the resultingproteins were analyzed by SDS-polyacrylamide gel electrophoresis andnative-gel electrophoresis. The analysis results are shown in FIGS. 3and 4.

As shown in FIGS. 3 and 4, which show the results of SDS-PAGE analysisof the wild-type flagellin (FlaB) and the mutated flagellin FlaB, thepoint-mutated flagellin proteins (D70A, R93A, L97W, N101A, S103W, E108A,N135D, A151W, N153W, V291W and D70A/N135D) of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20 and 44, and the wild-type flagellin (FlaB)protein, all showed a major band at 41.5 kDa. In the results ofnative-gel electrophoresis, it was observed that the wild-type FlaBprotein formed a huge multimer, so that most of the protein remained inthe stacking gel, and some of the FlaB protein, introduced into theresolving gel, was distributed between 66 kDa and 140 kDa. However, thepoint-mutated flagellin proteins (D70A, R93A, L97W, N101A, S103W, E108A,N135D, A151W, N153W, V291W and D70A/N135D) according to the presentinvention formed multimers at about 140 kDa. The above results suggestthat the wild-type FlaB protein formed a huge multimer in an aqueoussolution, but the point-mutated flagellin mutants (D70A, R93A, L97W,N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D)according to the present invention were present as multimers having asize smaller than that of the wild-type flagellin protein. Accordingly,it could be confirmed that the inventive flagellin mutants (D70A, R93A,L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) ofSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44 suppressed thepolymerization of flagellin.

Test Example 2 TLR5-Stimulating Activity of Flagellin Mutants

In order to measure the transcriptional activity of NF-κB, involved inTLR5-mediated signaling, so as to determine the TLR5-stimulatingactivities of the wild-type FlaB and the inventive flagellin mutants ofSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44, 293T cells weredispensed into each well of 24-well plates at a density of 1×10⁵cells/well and cultured overnight. Then, a reporter plasmid NF-κB-Luc(provided by professor Jeong-Mok Kim, Institute of Biomedical Science,Hanyang University College of Medicine, Korea) enabling transcriptionalactivity to be observed, a TLR5 gene-cloned p3xFlag-hTLR5 plasmid(provided by Dr. Steven B. Mizel, Departments of Microbiology andImmunology, Wake Forest University School of Medicine, USA) and abeta-galactosidase expression plasmid (Clontech) were simultaneouslyintroduced into the cells using FuGENE6 (Roche). After the cells wereadditionally cultured for 24 hours, the cultures were replaced withfresh media, and the cells were treated with the flagellin mutants(D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W andD70A/N135D) of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44,isolated in Example 1, for 18-24 hours. Then, the transcription of NF-κBin the cells was assayed by measuring the luciferase activity using aluminometer (Berthold), and the measurement results are shown in FIGS. 5and 6. As shown in FIGS. 5 and 6, the group treated with 20 ng/ml of thewild-type FlaB protein showed an increase in TLR5-mediated NF-κBtranscription activity of about 6.6 fold compared to the control grouptreated only with phosphate-buffered solution. However, the groupstreated with 20 ng/ml of each of the recombinant flagellin FlaB mutants(D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W andD70A/N135D) showed increases in TLR5-mediated NF-κB transcriptionactivity of about 24.6 fold, 14 fold, 25 fold, 22.6 fold, 23.7 fold, 21fold, 21.7 fold, 22.5 fold, 24.5 fold, 16.6 fold and 14.9 fold,respectively, compared to the control group treated only withphosphate-buffered solution.

[Sequence List Text]

SEQ ID NOS: 1 and 2 are the gene and amino acid sequences of a flagellinmutant (D70A), prepared by site-directed mutagenesis of aspartic acid(D70) to alanine (A) at position 70 of the V. vulnificus flagellin FlaB.

SEQ ID NOS: 3 and 4 are the gene and amino acid sequences of a flagellinmutant (R93A), prepared by site-directed mutagenesis of arginine (R93)to alanine (A) at position 93 of the V. vulnificus flagellin FlaB.

SEQ ID NOS: 5 and 6 are the gene and amino acid sequences of a flagellinmutant (L97W), prepared by site-directed mutagenesis of leucine (L97) totryptophan (W) at position 97 of the V. vulnificus flagellin FlaB.

SEQ ID NOS: 7 and 8 are the gene and amino acid sequences of a flagellinmutant (N101A), prepared by site-directed mutagenesis of asparagine(N101) to alanine (A) at position 101 of the V. vulnificus flagellinFlaB.

SEQ ID NOS: 9 and 10 are the gene and amino acid sequences of aflagellin mutant (S103W), prepared by site-directed mutagenesis ofserine (S103) to tryptophan (W) at position 103 of the V. vulnificusflagellin FlaB.

SEQ ID NOS: 11 and 12 are the gene and amino acid sequences of aflagellin mutant (E108A), prepared by site-directed mutagenesis ofglutamic acid (E108) to alanine (A) at position 108 of the V. vulnificusflagellin FlaB.

SEQ ID NOS: 13 and 14 are the gene and amino acid sequences of aflagellin mutant (N135D), prepared by site-directed mutagenesis ofasparagine (N135) to aspartic acid (D) at position 135 of the V.vulnificus flagellin FlaB.

SEQ ID NOS: 15 and 16 are the gene and amino acid sequences of aflagellin mutant (A151W), prepared by site-directed mutagenesis ofalanine (A151) to tryptophan (W) at position 151 of the V. vulnificusflagellin FlaB.

SEQ ID NOS: 17 and 18 are the gene and amino acid sequences of aflagellin mutant (N153W), prepared by site-directed mutagenesis ofasparagine (N153) to tryptophan (W) at position 153 of the V. vulnificusflagellin FlaB.

SEQ ID NOS: 19 and 20 are the gene and amino acid sequences of aflagellin mutant (V291W), prepared by site-directed mutagenesis ofvaline (V291) to tryptophan (W) at position 291 of the V. vulnificusflagellin FlaB.

SEQ ID NOS: 21 to 42 are primer sequences used in the flagellin mutantsof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44.

SEQ ID NOS: 43 and 44 are the gene and amino acid sequences of aflagellin mutant (D70A/N135D), prepared by site-directed mutagenesis ofaspartic acid (D70) to alanine (A) at position 70 of the V. vulnificusflagellin FlaB, and asparagine (N135) to aspartic acid (D) at position135 of the V. vulnificus flagellin FlaB.

1. A Vibrio vulnificus flagellin mutant having enhanced toll-likereceptor (TLR-5)-stimulating activity.
 2. The V. vulnificus flagellinmutant of claim 1, wherein the mutant is a flagellin mutant (D70A) ofSEQ ID NO: 2, prepared by site-directed mutagenesis of aspartic acid(D70) to alanine (A) at position 70 of the Vibrio vulnificus flagellinFlaB.
 3. The V. vulnificus flagellin mutant of claim 1, wherein themutant is a flagellin mutant (R93A) of SEQ ID NO: 4, prepared bysite-directed mutagenesis of arginine (R93) to alanine (A) at position93 of the V. vulnificus flagellin FlaB.
 4. The V. vulnificus flagellinmutant of claim 1, wherein the mutant is a flagellin mutant (L97W) ofSEQ ID NO: 6, prepared by site-directed mutagenesis of leucine (L97) totryptophan (W) at position 97 of the V. vulnificus flagellin FlaB. 5.The V. vulnificus flagellin mutant of claim 1, wherein the mutant is aflagellin mutant (N101A) of SEQ ID NO: 8, prepared by site-directedmutagenesis of asparagine (N101) to alanine (A) at position 101 of theV. vulnificus flagellin FlaB.
 6. The V. vulnificus flagellin mutant ofclaim 1, wherein the mutant is a flagellin mutant (S103W) of SEQ ID NO:10, prepared by site-directed mutagenesis of serine (S103) to tryptophan(W) at position 103 of the V. vulnificus flagellin FlaB.
 7. The V.vulnificus flagellin mutant of claim 1, wherein the mutant is aflagellin mutant (E108A) of SEQ ID NO: 12, prepared by site-directedmutagenesis of glutamic acid (E108) to alanine (A) at position 108 ofthe V. vulnificus flagellin FlaB.
 8. The V. vulnificus flagellin mutantof claim 1, wherein the mutant is a flagellin mutant (N135D) of SEQ IDNO: 14, prepared by site-directed mutagenesis of asparagine (N135) toaspartic acid (D) at position 135 of the V. vulnificus flagellin FlaB.9. The V. vulnificus flagellin mutant of claim 1, wherein the mutant isa flagellin mutant (A151W) of SEQ ID NO: 16, prepared by site-directedmutagenesis of alanine (A151) to tryptophan (W) at position 151 of theV. vulnificus flagellin FlaB.
 10. The V. vulnificus flagellin mutant ofclaim 1, wherein the mutant is a flagellin mutant (N153W) of SEQ ID NO:18, prepared by site-directed mutagenesis of aspargine (N153) totryptophan (W) at position 153 of the V. vulnificus flagellin FlaB. 11.The V. vulnificus flagellin mutant of claim 1, wherein the mutant is aflagellin mutant (V291W) of SEQ ID NO: 20, prepared by site-directedmutagenesis of valine (V291) to tryptophan (W) at position 291 of the V.vulnificus flagellin FlaB.
 12. The V. vulnificus flagellin mutant ofclaim 1, wherein the mutant is a flagellin mutant (D70A/N135D) of SEQ IDNO: 44, prepared by site-directed mutagenesis of aspartic acid (D70) toalanine (A) at position 70 of the V. vulnificus flagellin FlaB, andasparagine (N135) to aspartic acid (D) at position 135 of the V.vulnificus flagellin FlaB.
 13. A vaccine adjuvant containing, as anactive ingredient, at least one flagellin mutant selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and
 44. 14.Use of at least one flagellin mutant selected from the group consistingof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44, forstimulating the activity of a toll-like receptor-5 (TLR5).